Methods of improving the introduction of DNA into bacterial cells

Abstract

The present invention relates to methods of improving the introduction of DNA into bacterial host cells.

1. An isolated polynucleotide encoding a DNA methyltransferase selected from:
 (a) a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 95% sequence identity with amino acids 1 to 337 of SEQ ID NO: 2; 
 (b) a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity with nucleotides 1 to 1011 of SEQ ID NO: 1; and 
 (c) a polynucleotide that hybridizes under high stringency conditions with nucleotides 1 to 1011 of SEQ ID NO: 1 or its full-length complementary strand wherein said high stringency conditions are hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C. 
 
     
     
       2. The isolated polynucleotide of  claim 1 , which encodes a DNA methyltransferase comprising an amino acid sequence having at least 95% sequence identity with amino acids 1 to 337 of SEQ ID NO: 2. 
     
     
       3. The isolated polynucleotide of  claim 1 , which encodes a DNA methyltransferase comprising an amino acid sequence having at least 97% sequence identity with amino acids 1 to 337 of SEQ ID NO: 2. 
     
     
       4. The isolated polynucleotide of  claim 1 , which encodes a DNA methyltransferase comprising an amino acid sequence having at least 99% sequence identity with amino acids 1 to 337 of SEQ ID NO: 2. 
     
     
       5. The isolated polynucleotide of  claim 1 , which encodes a DNA methyltransferase comprising or consisting of amino acids 1 to 337 of SEQ ID NO: 2. 
     
     
       6. The isolated polynucleotide of  claim 1 , comprising a nucleotide sequence having at least 95% sequence identity with nucleotides 1 to 1011 of SEQ ID NO: 1. 
     
     
       7. The isolated polynucleotide of  claim 1 , comprising a nucleotide sequence having at least 97% sequence identity with nucleotides 1 to 1011 of SEQ ID NO: 1. 
     
     
       8. The isolated polynucleotide of  claim 1 , comprising a nucleotide sequence having at least 99% sequence identity with nucleotides 1 to 1011 of SEQ ID NO: 1. 
     
     
       9. The isolated polynucleotide of  claim 1 , comprising nucleotides 1 to 1011 of SEQ ID NO: 1. 
     
     
       10. The isolated polynucleotide of  claim 1 , comprising the DNA methyltransferase coding sequence contained in plasmid pMDT138 which is contained in  Escherichia coli  NRRL B-41967. 
     
     
       11. The isolated polynucleotide of  claim 1 , wherein the polynucleotide hybridizes under high stringency conditions with nucleotides 1 to 1011 of SEQ ID NO: 1 or its full-length complementary strand wherein said high stringency conditions are hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C. 
     
     
       12. The isolated polynucleotide of  claim 1 , wherein the polynucleotide hybridizes under very high stringency conditions with nucleotides 1 to 1011 of SEQ ID NO: 1 or its full-length complementary strand wherein said high stringency conditions are hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C. 
     
     
       13. A nucleic acid construct or recombinant expression vector comprising the polynucleotide of  claim 1  operably linked to one or more control sequences that direct the production of the DNA methyltransferase in an expression host. 
     
     
       14. The nucleic acid construct or recombinant expression vector of  claim 13 , wherein the polynucleotide encodes a DNA methyltransferase comprising an amino acid sequence having at least 95% sequence identity with amino acids 1 to 337 of SEQ ID NO: 2. 
     
     
       15. The nucleic acid construct or recombinant expression vector of  claim 13 , wherein the polynucleotide encodes a DNA methyltransferase comprising or consisting of amino acids 1 to 337 of SEQ ID NO: 2. 
     
     
       16. A recombinant host cell comprising the polynucleotide of  claim 1 . 
     
     
       17. The recombinant host cell of  claim 16 , wherein the polynucleotide encodes a DNA methyltransferase comprising an amino acid sequence having at least 95% sequence identity with amino acids 1 to 337 of SEQ ID NO: 2. 
     
     
       18. The recombinant host cell of  claim 16 , wherein the polynucleotide encodes a DNA methyltransferase comprising or consisting of amino acids 1 to 337 of SEQ ID NO: 2. 
     
     
       19. A method of producing a DNA methyltransferase, comprising: (a) cultivating the recombinant host cell of  claim 16  under conditions conducive for production of the DNA methyltransferase; and (b) recovering the DNA methyltransferase. 
     
     
       20. A method of producing bacterial transformants, comprising:
 (a) introducing a DNA into a first bacterial host cell comprising the polynucleotide of  claim 1  encoding a DNA methyltransferase to methylate the DNA; 
 (b) transferring the methylated DNA from the first bacterial host cell into a second bacterial host cell, wherein the second bacterial host cell comprises a restriction endonuclease able to degrade the DNA but unable to degrade the methylated DNA; and 
 (c) isolating transformants of the second bacterial host cell comprising the methylated DNA. 
 
     
     
       21. The method of  claim 20 , wherein the polynucleotide encodes a DNA methyltransferase comprising an amino acid sequence having at least 95% sequence identity with amino acids 1 to 337 of SEQ ID NO: 2. 
     
     
       22. The method of  claim 20 , wherein the polynucleotide encodes a DNA methyltransferase comprising or consisting of amino acids 1 to 337 of SEQ ID NO: 2.